Surgery – Reproduction and fertilization techniques
Reexamination Certificate
2000-11-22
2003-04-08
Hindenburg, Max F. (Department: 3736)
Surgery
Reproduction and fertilization techniques
Reexamination Certificate
active
06544166
ABSTRACT:
The present invention related to a laboratory procedure and a pharmaceutical composition and their use to treat human infertility. More specifically, this invention relates to the treatment of human infertility by in vitro methods that actively arrest the human oocyte in prophase of first meiotic division and subsequently actively reverse this meiotic blockage by either adding at least one meiosis-stimulating compound or by culture conditions leading to endogenous formation of meiotic stimulating molecules in the oocyte-cumulus complex. Briefly, this invention relates to an improved method of in vitro fertilization (hereinafter designated IVF).
BACKGROUND OF THE INVENTION
Meiosis is the unique and ultimate event that occurs to germ cells and on which sexual reproduction is based. Meiosis comprises two meiotic divisions. During the first division, exchange between maternal and paternal genes takes place before the pairs of chromosomes are separated into each of the two daughter cells. These contain only half the number (1n) of chromosomes and 2c DNA. The second meiotic division proceeds without DNA synthesis. This division therefore results in the formation of the haploid germ cells with only 1c DNA.
The meiotic events are similar in male and female germ cells, but the time schedule and the differentiation processes which lead to the formation of ova and spermatozoa differ profoundly. All female germ cells enter the prophase of the first meiotic division early in life, often before birth, but all are arrested as oocytes later in the prophase (dictyate state) until ovulation after puberty. Thus, from early life the female has a stock of oocytes, which is drawn upon until the stock is exhausted. Meiosis in oocytes is not completed until after fertilization, and results in only one ovum and two abortive polar bodies per germ cell. In contrast, only some of the male germ cells enter meiosis from puberty and leave a stem cekk population of germ cells throughout life. Once initiated, meiosis in the male cell proceeds without significant delay and produces 4 spermatozoa.
Little is known about the mechanisms that control meiosis in the male and in the female. In the oocyte, recent studies indicate that follicular purines such as hypoxanthine and adenosine could be responsible for meiotic arrest (Downs, S. M., et al. (1985) in Dev. Biol. 82: 454-458; Eppig, J. J., et al. (1986) in Dev. Biol. 119: 313-321; and Downs, S. M., et al. (1993) in Mol. Reprod. Dev. 35: 82-94). These purine bases were found in follicular fluid in millimolar concentrations (Eppig, J. J., et al. (1985) in Biol. Reprod. 33: 1041-1049). However, the purine base-induced arrest was reversible. This was demonstrated by experiments in which mouse and human oocytes were maintained in meiotic arrest for 24 hours with hypoxanthine followed by a 16-30 hour culture in inhibitor-free medium (Downs, S. M., et al. (1986) in Gamet Res. 15: 305-316; and Cha, K. Y., et al. (1992) in Reprod. Fertil. Dev. 4: 695-701). Nearly 100% of the arrested mouse oocytes resumed maturation and, furthermore, the mature oocytes were successfully fertilized and demonstrated complete pre- and post-implantation development. These data collectively support the idea that purines such as hypoxanthine and adenosine are physiologically important in the mechanisms controlling meiotic arrest in vivo.
Cyclic adenosine 5′-monophosphate (hereinafter designated cAMP) plays a pivotal role as a second messenger in the signal transduction pathway during meiosis in the oocyte. cAMP is generated by the action of adenylate cyclase (hereinafter designated AC). cAMP is degraded by the family of phosphodiesterase enzymes (hereinafter designated PDE), which produces inactive second messenger products. Hypoxanthine (hereinafter designated Hx) is an inhibitor of cAMP PDE (Eppig, J. J., et al. (1985) in Biol. Reprod. 33: 1041-1049). As such, Hx can prevent the hydrolysis of oocyte cAMP and thereby maintain elevated levels of cAMP in the oocyte. In addition to hypoxanthine, agents acting upstream or downstream of cAMP are able to increase cAMP levels. By this mechanism, activation of AC with forskolin, inhibition of PDE with the nonselective 3-isobutyl-1-methylxanthine (hereinafter designated IBMX), or inhibition of the oocyte-specific isoform PDE3 with a specific PDE3-inhibitor, for example milrinone, leads to meiotic arrest by maintaining elevated levels of c-AMP within the oocytes (Downs, S. M., and Hunzicker-Dunn, M., (1995) in Dev Biol 172: 72-85; and Tsafiri, A., et al. (1996) in Dev Biol 178: 393-402).
A PDE3 specific inhibitor has been described as a contraceptive agent (WO98/10765).
The presence of a diffusible meiosis regulating substance was first described (Byskov, A. G., et al. (1976) in Dev. Biol. 52: 193-200) in foetal mouse gonads. A meiosis activating substance (MAS) was secreted in the foetal mouse ovaries in which meiosis was ongoing, and a meiosis preventing substance (hereinafter designated MPS) was released from morphologically differentiated testes containing resting, non-meiotic germ cells. It was, therefore, suggested that the relative concentrations of MAS and MPS regulated theinitiation, arrest, and resumption of meiosis in male and female germ cells (Byskov, A. G., and Hoyer, P. E., (1994) in The Physiology of Reproduction, Knobil, E., and Neill, J. D., (Editors), Raven Press, New York, pp. 487-540). A recent article (Byskov, A. G., et al. (1995) in Nature 374: 559-562) describes the isolation of certain sterols from preovulatory ovarian follicular fluid, defined as FF-MAS, and bull testicular testis, defined as T-MAS, that activate oocyte meiosis. This was confirmed recently (Grøndahl et al. (1998) in Biol. Reprod. 1998; 58:1297-1302) by showing that de novo synthesized FF-MAS is capable of mediating resumption of meiosis in mice oocytes.
Since the first IVF baby was delivered in 1978, this procedure has resulted in thousands of pregnancies and opened a vast new frontier of research and treatment for infertile couples. Still, there is a significant need for improved infertility treatment modalities today. It is presumed that about one out of seven couples experience problems with sub-fertility or infertility.
IVF of human oocytes has become commonly used for the treatment of female and male sub-fertility. The standard IVF treatment includes a long phase of hormone stimulation of the female patient, for example 30 days, which is initiated by suppressing the patient+s own follicle stimulating hormone (hereinafter designated FSH) and luteinizing hormone (hereinafter designated LH) by gonadotropin releasing hormone (hereinafter designated GnRH), and this is followed by injections of exogenous gonadotropins, for example FSH and/or LH, in order to ensure development of multiple preovulatory follicles and aspiration of multiple in vivo matured oocytes immediately before ovulation. The aspirated oocyte is subsequently fertilized in vitro and cultured, typically for three days, before transfer back into the uterus at the 4-8 cell stage. Continuous efforts have been made to optimise and simplify this procedure. Nevertheless, the overall pregnancy rate cannot be increased significantly over about 20% with the current treatment modalities. In a large European survey of IVF patients, it was found that 7.2 oocytes out of 11.5 aspirated oocytes per patient had undergone resumption of meiosis immediately before fertilization, only 4.3 oocytes were fertilized and only 2.2 oocytes reached the 8-cell embryo stage after fertilization and in vitro culture (ESHRE, Edinburgh, 1997).
Due to the very unpredictable quality of the state of the art embryos today, more than one embryo has to be transferred just to give a reasonable chance of success. Therefore, it is common to transfer 2-3 embryos (up to 5 embryos in some countries), which carries the very large side effect of multiple pregnancies with great discomfort and risk to both patient and children. Moreover, it has been estimated that the increased health care expense due to multiple birth (twins, tripl
Cadugan Joseph A.
Green Reza
Hindenburg Max F.
LandOfFree
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